[Safety and efficacy of the early administration of levosimendan in patients with acute non-ST-segment elevation myocardial infarction and elevated NT-proBNP levels: An Early Management Strategy of Acute Heart Failure (EMS-AHF)].

Objectives: To investigated the safety and efficacy of treating patients with acute non-ST-segment elevation myocardial infarction (NSTEMI) and elevated levels of N-terminal pro-hormone B-type natriuretic peptide (NT-proBNP) with levosimendan within 24 hours of first medical contact (FMC). Methods: This multicenter, open-label, block-randomized controlled trial (NCT03189901) investigated the safety and efficacy of levosimendan as an early management strategy of acute heart failure (EMS-AHF) for patients with NSTEMI and high NT-proBNP levels. This study included 255 patients with NSTEMI and elevated NT-proBNP levels, including 142 males and 113 females with a median age of 65 (58-70) years, and were admitted in the emergency or outpatient departments at 14 medical centers in China between October 2017 and October 2021. The patients were randomly divided into a levosimendan group (n=129) and a control group (n=126). The primary outcome measure was NT-proBNP levels on day 3 of treatment and changes in the NT-proBNP levels from baseline on day 5 after randomization. The secondary outcome measures included the proportion of patients with more than 30% reduction in NT-proBNP levels from baseline, major adverse cardiovascular events (MACE) during hospitalization and at 6 months after hospitalization, safety during the treatment, and health economics indices. The measurement data parameters between groups were compared using the t-test or the non-parametric test. The count data parameters were compared between groups using the χ² test. Results: On day 3, the NT-proBNP levels in the levosimendan group were lower than the control group but were statistically insignificant [866 (455, 1 960) vs. 1 118 (459, 2 417) ng/L, Z=-1.25,P=0.21]. However, on day 5, changes in the NT-proBNP levels from baseline in the levosimendan group were significantly higher than the control group [67.6% (33.8%,82.5%)vs.54.8% (7.3%,77.9%), Z=-2.14, P=0.03]. There were no significant differences in the proportion of patients with more than 30% reduction in the NT-proBNP levels on day 5 between the levosimendan and the control groups [77.5% (100/129) vs. 69.0% (87/126), χ²=2.34, P=0.13]. Furthermore, incidences of MACE did not show any significant differences between the two groups during hospitalization [4.7% (6/129) vs. 7.1% (9/126), χ²=0.72, P=0.40] and at 6 months [14.7% (19/129) vs. 12.7% (16/126), χ²=0.22, P=0.64]. Four cardiac deaths were reported in the control group during hospitalization [0 (0/129) vs. 3.2% (4/126), P=0.06]. However, 6-month survival rates were comparable between the two groups (log-rank test, P=0.18). Moreover, adverse events or serious adverse events such as shock, ventricular fibrillation, and ventricular tachycardia were not reported in both the groups during levosimendan treatment (days 0-1). The total cost of hospitalization [34 591.00(15 527.46,59 324.80) vs. 37 144.65(16 066.90,63 919.00)yuan, Z=-0.26, P=0.80] and the total length of hospitalization [9 (8, 12) vs. 10 (7, 13) days, Z=0.72, P=0.72] were lower for patients in the levosimendan group compared to those in the control group, but did not show statistically significant differences. Conclusions: Early administration of levosimendan reduced NT-proBNP levels in NSTEMI patients with elevated NT-proBNP and did not increase the total cost and length of hospitalization, but did not significantly improve MACE during hospitalization or at 6 months.

Important aspects of Toll-like receptors, ligands and their signaling pathways.

Due to the rapid increase of new information on the multiple roles of Toll-like receptors (TLRs), this paper reviews several main properties of TLRs and their ligands and signaling pathways. The investigation of pathogen infections in knockout mice suggests that specific TLRs play a key role in the activation of immune responses. Although the investigation of TLR biology is just beginning, a number of important findings are emerging. This review focuses on the following seven aspects of this emerging field: (a) a history of TLR and ligand studies; (b) the molecular basis of recognition by TLRs: TLR structures, pathogen-associated molecular pattern binding sites, TLR locations and functional responses; (c) cell types in TLR expression; (d) an overview of TLRs and their ligands: expression and ligands of cell-surface TLRs and of intracellular TLRs; (e) TLR-signaling pathways; (f) discussion: TLRs control of innate and adaptive systems; the trafficking of intracellular TLRs to endolysosomes; investigation of TLRs in regulating microRNA; investigation of crystal structure of TLRs with ligand binding; incidence of infectious diseases associated with single nucleotide polymorphisms (SNPs) in TLR genes; risk of cancer related to SNPs in TLR genes; TLR-ligand mediated anti-cancer effects; and TLR-ligand induced chronic inflammation and tumorigenesis; and (g) conclusions.

Protocol optimization for long-term liquid storage of goat semen in a chemically defined extender.

A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of spermatozoa in extenders containing egg yolk or milk. The use of chemically defined extenders will have obvious advantages in liquid storage of buck semen. Our previous study showed that the self-made mZAP extender performed better than commercial extenders, and maintained a sperm motility of 34% for 9 days and a fertilizing potential for successful pregnancies for 7 days. The aim of this study was to extend the viability and fertilizing potential of liquid-stored goat spermatozoa by optimizing procedures for semen processing and storage in the mZAP extender. Semen samples collected from five goat bucks of the Lubei White and Boer breeds were diluted with the extender, cooled and stored at 5 degrees C. Stored semen was evaluated for sperm viability parameters, every 48 h of storage. Data from three ejaculates of different bucks were analysed for each treatment. The percentage data were arcsine-transformed before being analysed with anova and Duncan's multiple comparison test. While cooling at the rate of 0.1-0.25 degrees C/min did not affect sperm viability parameters, doing so at the rate of 0.6 degrees C/min from 30 to 15 degrees C reduced goat sperm motility and membrane integrity. Sperm motility and membrane integrity were significantly higher in semen coated with the extender containing 20% egg yolk than in non-coated semen. Sperm motility, membrane integrity and acrosomal intactness were significantly higher when coated semen was 21-fold diluted than when it was 11- or 51-fold diluted and when extender was renewed at 48-h intervals than when it was not renewed during storage. When goat semen coated with the egg yolk-containing extender was 21-fold diluted, cooled at the rate of 0.07-0.25 degrees C/min, stored at 5 degrees C and the extender renewed every 48 h, a sperm motility of 48% was maintained for 13 days, and an in vitro-fertilizing potential similar to that of fresh semen was maintained for 11 days.

Liquid storage of goat semen in chemically defined extenders.

The suitability of certain commercial and self-made chemically defined extenders for liquid storage of goat semen was tested and the effects of storage temperatures, dilution rates and sperm washing and pH of extenders on the goat sperm during liquid storage were observed. Semen was collected from nine goat bucks of the Lubei White and Boer breeds using an artificial vagina. Each ejaculate after initial evaluation was diluted with a specific extender, cooled and stored at a desired temperature. Stored semen was evaluated for sperm motility and other parameters every 24 or 48 h of storage. The ranking order of the existing milk- and yolk-free extenders in sustaining goat sperm motility was Androhep > Zorlesco > Beltsville thawing solution > the Tris-glucose medium. The new extender (mZA) which was formulated based on Zorlesco and Androhep was more suitable for goat sperm than Androhep. The mZAP extender with Bovine Serum Albumin (BSA) replaced with polyvinyl alcohol (PVA) worked as efficiently as the mZA in maintaining sperm motility, membrane integrity, acrosome intactness and capacitation status. Goat sperm motility was best maintained at 5 degrees C during liquid preservation, but decreased significantly as the temperature increased. When semen was sixfold diluted, sperm motility was maintained longer (p < 0.05) after centrifugation, but sperm motility did not differ between the centrifuged and non-centrifuged groups when semen was 11-fold diluted. When the extender pH was adjusted from 6.6 to 6.04, the efficiency increased significantly in both Androhep and mZAP. A forward sperm motility of 34% was maintained for 9 days when buck semen was 11-fold diluted and stored at 5 degrees C in mZAP, with pH adjusted to 6.04. It is concluded that for liquid storage of buck semen, the mZA extender was more suitable than other extenders; BSA can be replaced with PVA in mZA; centrifugation to remove seminal plasma can be omitted by adequate dilution; and the storage temperature and pH of extenders affected sperm motility significantly.

Interferon-induced differentiation of U937 cells. Comparison with other agents that promote differentiation of human myeloid or monocytelike cell lines.

Effects of human fibroblast (beta) or leukocyte (alpha) interferon (IFN) on differentiations of a human histiocytic lymphoma-derived cell line (U937) or promyelocytic leukemia-derived cell line (HL-60) were studied. When cultured with beta-IFN (400-1,000 U/ml), U937 cells showed gross morphologic and microscopic changes consisting of clumping, increased cytoplasmic-to-nuclear ratio, enhanced prominence of cytoplasmic granules, and membrane ruffling. After culture with beta-IFN, the number of U937 cells reactive with B43.4.1 monoclonal antibody, which is specific for human monocytes, natural killer cells, and neutrophils, increased from less than 10% of U937 cells to 47% beta-IFN treatment also enhanced antibody-dependent cellular cytotoxicity against chicken erythrocytes by U937 cells. The same morphologic, phenotypic, and functional changes were also observed when U937 were treated with recombinant or natural alpha-IFN. The effects of alpha-IFN were totally abolished by anti-alpha-IFN serum. In contrast, HL-60, which differentiates toward cells of the monocyte lineage in response to phorbol 12-myristate 13-acetate (based on the above criteria), and toward granulocytes in response to dimethyl sulfoxide, did not differentiate when cultured with alpha- or beta-IFN. No consistent relationship between induction of differentiation and changes in phospholipid methylation were observed.

Effects of follicular atresia and size on the developmental competence of bovine oocytes: a study using the well-in-drop culture system.

The effect of granulosa cell (GC) apoptosis and follicle size on the competence of bovine oocytes were studied using a well-in-drop (WID) oocyte/embryo culture system, which allows identification of follicular origin. Hatching rates of blastocysts did not differ (P>0.05) between oocytes cultured in the WID system (13%) and those cultured in the conventional group system (16%). Hatching rates of blastocysts were higher (P<0.05) in early atretic (17%) than in non-atretic (8%) and late atretic follicles (10%) of the same size (4-8mm), and in 6-8mm (22%) than in 4-5mm follicles (15%) at the early atretic stage. More oocytes (P<0.05) from late atretic (17%) than from non-atreteic (7%) or early atretic follicles (9%) of the same size (4-8mm) were arrested at Grade 1 cumulus expansion (only cells in the peripheral two layers began to expand). Similarly, more oocytes from 2 to 3mm follicles (30%) than from 6 to 8mm follicles (21%) at the same (late) atretic stage had Grade 1 cumulus expansion (P<0.05). Hatching blastocyst percentages of oocytes with Grade 3 (all layers of the cumulus except corona radiate cells expanded) or Grade 4 (full) cumulus expansion were higher in early atretic (20%) than in non-atretic (13%) or late atretic follicles (12%). Hatching blastocyst percentages of oocytes from follicles at the early atretic stage increased as cumulus expanded from Grade 2 (9%) to Grade 4 (27%). Regardless of the degree of follicle atresia, 72-76% of the floating cells in the follicular fluid (FF) were undergoing apoptosis. The floating cell density in FF was highly (r=0.6-0.7) correlated with oocyte developmental potency. In conclusion, the WID culture system was as efficient as group culture and allowed identification of follicular origin. Furthermore, the developmental potential of oocytes was affected by GC apoptosis, follicle size and cumulus expansion, and the floating cell density in FF could be used as a simple and non-invasive marker of oocyte quality.

Fibronectin cell-binding domain triggered transmembrane signal transduction in human monocytes.

Fibronectin (Fn) fragments have recently been shown to stimulate tumor necrosis factor (TNF) secretion by human monocytes. In this study, we investigated the signal transduction mechanisms involved in Fn-induced TNF secretion. Treatment of human monocytes with Fn120, a chymotryptic cell-binding fragment of plasma Fn, failed to cause a detectable rise in Ca2+ mobilization. Fn120-induced TNF secretion could be inhibited with Ca2+ channel blockers. The protein kinase C (PKC) inhibitors H-7 and sphingosine inhibited the TNF-inducing activity of Fn120. HA1004 was used as a control for the isoquinoline sulfonamide derivatives and did not change Fn120-induced TNF secretion by monocytes. H-8 inhibited TNF secretion at higher concentrations. A calmodulin-dependent kinase inhibitor, W-7, was found to be effective, with 50% inhibition of Fn120-induced TNF secretion at 5 microM. The activation and translocation of PKC were measured directly. In unstimulated monocytes, approximately 70% of PKC activity was found in the cytosol and 30% in the membrane. Following the stimulation of monocytes with phorbol myristate acetate (100 nM), rapid and sustained translocation of PKC from the cytosol to the membrane was observed. The stimulation of monocytes with Fn120 triggered a rapid translocation of PKC within 2 to 5 min, followed by a return to normal levels within 8 min. These findings support the conclusion that Fn120-induced TNF secretion requires the activation of PKC.

Expression of cytokine mRNA during immuno-modulation of murine suppressor macrophages.

In order to analyze the mechanism of immuno-modulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay, checked the changes of mRNA expression pattern of several cytokine genes during the immuno-modulation. It has been found that, after treating peritoneal suppressor macrophages with LPS, mRNAs of IL-12 p35, IL-12 p40, IL-6 and IFN-gamma are newly appeared, while those of IL-1 alpha, IL-1 beta and IL-1Ra are increased and those of other cytokines, like TGF-beta 1 and MIF are not changed at all. It seems certain that those cytokines, whose expression is increased by LPS stimulation, may be responsible for the functional changes of suppressor macrophages during immuno-modulation. Among these changes, the appearance of IL-12 mRNA may play a critical role, and, in this regard, the synergetic effect between IFN-gamma and LPS on the increase of IL-12 p35 and Il-12 p40 mRNA expression is an interesting finding.

Influence of acute and chronic ethanol treatment on muscarinic responses and receptor expression in Chinese hamster ovary cells.

The influence of ethanol on the muscarinic receptor-mediated release of inositol phosphate from Chinese hamster ovary (CHO) cells stably transfected with one of the five subtypes of muscarinic acetylcholine receptor was determined. In CHO cells expressing M3 muscarinic receptors (CHO-M3), carbamylcholine increased muscarinic receptor-induced release of inositol phosphate by 150-350% following a 15-min incubation with an EC50 of approximately 30 microM. Maximal responses were obtained with 1 mM carbamylcholine, while responses to 10 mM carbamylcholine were somewhat less than maximal. Preincubation with atropine for 10 min inhibited the response with an IC50 of approximately 30 nM. CHO cells transfected with M1, M3, and M5 receptors displayed a similar pattern of activity; CHO cells transfected with M2 and M4, as well as untransfected cells, were unresponsive to carbamylcholine. Ethanol acutely inhibited the response of CHO-M3 cells to carbamylcholine by 15% at 18 mM and by 47% at 180 mM (the highest concentration examined). CHO-M3 cells were incubated with 50 mM ethanol for 48 hr. This treatment did not affect the number of cells or their protein content (113 pg/cell). The expression of M3 muscarinic receptors (determined using [3H]N-methylscopolamine) increased from 1.34 +/- 0.23 to 1.75 +/- 0.16 pmol/mg protein (P < 0.05). In contrast, carbamylcholine-stimulated release of inositol phosphate was depressed by 40-70% in four experiments. Concentration-response analyses indicated a non-competitive inhibitory mechanism. This dissociation of muscarinic receptor expression and muscarinic signaling suggests a compensatory increase in receptor expression in response to chronic inhibition of muscarinic signaling by ethanol.

Phospholipase A2 activity in peripheral blood cells of rheumatoid arthritis patients treated with methotrexate: a preliminary study.

OBJECTIVE: To measure the activity of phospholipase A2 (PLA2) in peripheral blood mononuclear cells (PBMC), polymorphonuclear leukocytes (PMN) and platelets in patients with rheumatoid arthritis (RA) before and during treatment with methotrexate (MTX). METHODS: Nine patients with RA treated with MTX were studied for six months. PBMC, PMN, and platelets were isolated at baseline and at monthly intervals thereafter. PLA2 activity was measured in lysates of the various cell fractions by the hydrolysis of radiolabeled lysocholine from phosphatidylcholine, L-Dipalmitoyl using thin layer chromatography. Data were calculated as pmol of 14C choline released per hour per milligram of cellular protein. RESULTS: At baseline, compared to normal controls, PLA2 activity was significantly increased in platelets (p = 0.02) but not in PBMC (p = 0.32) nor in PMN (p = 0.23). Seven of the nine patients responded clinically to treatment with MTX. In these 7 patients over the six-month treatment course, there was a significant decrease in PLA2 activity in the platelets which correlated with improvement in disease activity (R = 0.82, p = 0.03). PLA2 activity also decreased in PBMC and PMN, but the correlation with disease improvement was not statistically significant (R = 0.71, p = 0.07 for PBMC; and R = 0.43, p = 0.33 for PMN). CONCLUSION: PLA2 activity in platelets but not in PBMC or PMN is significantly increased in patients with RA compared to normal controls. Although PLA2 activity decreased in all the cells during treatment with MTX, only PLA2 activity in platelets correlated significantly with improvement in disease activity.

Simple and rapid determination of a new pharmaceutically active amine hydrochloride.

The quantitative analysis of a new pharmaceutically active amine hydrochloride is described. Samples are extracted with chloroform. A yellow amine-dye complex is formed by buffering a sample-bromthymol blue solution at pH 8.5 +/- 0.1 and subsequently extracted with chloroform. The complex is treated with 0.01 N NaOH to convert it back to the sodium salt of bromthymol blue, which is then measured at 615 nm in the aqueous layer. The amount of complex extracted is linearly related to the amount of amine present, from 0.020 to 0.20 mg/ml. Under the selected conditions, the compound can be determined in the presence of degradation products and there is no interference from common pharmaceutical excipients. The method is suitable for stability studies.

[A new signal receptor family--a review of the current progress of Toll/IL-1R family].

Toll receptor was first identified in studies of dorsalventral polarity formation of drosophila embryo. As its similarity in structure, function and signal transduction pathway to the IL-1 receptor, Toll becomes a member of a new signal receptor family--the Toll/IL-1R family. Besides participating in embryonic development, this signal receptor family also plays a key role in triggering innate defenses against pathogens, in which the function of human Toll protein in the pathogenesis of LPS is especially interested.

The treatment of severe and multiple injuries in intensive care unit: report of 80 cases.

OBJECTIVE: To summarize our case load in managing severe and multiple injuries (SMI) in the Intensive Care Unit (ICU). PATIENTS AND METHODS: The clinical data of 80 SMI patients treated in our ICU from January 2009 to June 2013 were analyzed. RESULTS: Results of these 80 SMI patients, 60 (75%) were salvaged and 15 (18.75%) died. The causes of death included severe head injury (n=7), severe chest injury (n=3), destruction of injured abdominal organs (n=2), and multiple organ dysfunction syndrome (n=3). Five patients (7.50%) gave up treatment and were discharged upon their own requests. Early application of continuous renal replacement therapy (CRRT) and enteral nutrition (EN) improved outcomes. CONCLUSIONS: The key interventions during the ICU treatment of SMI include: adequate analgesia and appropriate sedation; timely management of hypoxemia; reasonable fluid resuscitation and CRRT.

Protein kinase C activation in human monocytes: regulation of PKC isoforms.

The distribution of protein kinase C (PKC) isoforms and phorbol 12-myristate 13-acetate (PMA)-induced activation of PKC in human monocytes was investigated. Using Western blot analysis, PKC beta was found to be the most abundant isoform in monocytes. PKC beta was equally distributed in the cytosol and membrane. PKC-alpha was readily detectable and found predominantly in the cytosol. Little to no PKC-epsilon, gamma, delta, and zeta were observed. Following the treatment of monocytes with PMA, the physical translocation of PKC alpha from the cytosol to the membrane occurred over 60 min. PMA-induced translocation of PKC-beta was difficult to detect by Western blot. Fura-2 analysis demonstrated that PMA-induced PKC translocation was not accompanied by a net change in cytosolic calcium levels. Using histone as a substrate for PKC activity, an extremely rapid translocation of PKC-dependent histone phosphorylation (PKC-DHP) was induced by PMA. Cytosolic PKC-DHP activity decreased to undetectable levels within 8 min. In contrast, analysis of PKC-dependent endogenous substrate phosphorylation (PKC-DESP) showed a pattern with a time-course similar to that observed with Western blot. Thus, translocation of PKC-DESP but not PKC-DHP activity correlated with PKC-alpha as determined by Western blot. The data support the concept that PKC activity is substrate dependent and suggest that using one assay for the measurement of PKC activity may lead to erroneous conclusions.

[LPS and PMA induced PKC-alpha and PKC-epsilon activation and translocation in murine peritoneal macrophages].

Suppressor macrophages induced by the continuous invasion of tumor cells and parasites, which can acquire an ability in vitro to kill or inhibit tumor cells and inhibit the activity of T, B lymphocytes and NK cells, have been indicated. We have developed a procedure previously to modulate the suppressor macrophages by bacterial lipopolysaccharide (LPS). The modulated macrophages remained and even enhanced the ability to inhibit tumor growth and to up-regulate or enhance the activities of T, B lymphocytes and NK cells in vitro. However, the mechanisms of macrophage modulation by LPS are unknown. This investigation was designed to analyze the regulation of PKC activity and to characterize the isoforms of PKC during macrophage modulation by using Western blot and endogenous substrate phosphorylation (PKC-DESP). In rest cells, PKC-beta was found to be the most abundant isoform in macrophages; and PKC-alpha, beta was found predominantly in the cytosol. Using PMA as a positive control, we found that the immuno-modulator agent--LPS triggered the physical translocation from the cytosol onto the membrane of PKC-alpha and PKC-epsilon, but PKC-beta (beta I or beta II) was difficult to detect. The analysis of PKC-DESP showed a pattern with a time course similar to that observed with Western blot. We observed that LPS and PMA increase the level of phosphorylation of 55 kDa and 74 kDa proteins with a corresponding decrease in the cytosolic proteins. It suggests that the translocation of PKC-alpha and PKC-epsilon, may be important events involving in the PKC-pathway by LPS-mediated modulation in suppressor macrophages.

Protein kinase C in tumoricidal activation of mouse macrophage cell lines.

A potential role of protein kinase C (PKC) in lipopolysaccharide- (LPS-) induced tumoricidal activation of macrophages was investigated by using two mouse macrophage cell lines (P388D1 and J774). J774 cells are stimulated by LPS to kill target P815 mastocytoma cells, whereas P388D1 cells fail to develop such an ability. Pretreatment of J774 cells with H-7 or phorbol myristate acetate resulted in a significant inhibition of LPS-induced cytotoxicity, whereas pretreatment with H-8, ML-7, HA1004, or W-7 did not. Since these results suggested a critical role of PKC in the activation process, the properties of PKC in the two cell lines were compared. Western blotting with rabbit antiserum specific for the PKC beta regulatory domain allowed detection of a protein of 79 kilodaltons (kDa) in the detergent lysates of both cell lines that were not stimulated by LPS. However, LPS treatment resulted in the appearance of a second protein of 40 kDa only in J774 cells and not in P388D1 cells. Furthermore, two forms of protein kinase (one basic and the other acidic) were identified in the cytosol of J774 cells by HPLC on DEAE-5PW, whereas only the basic form was found in P388D1 cells. On the basis of the response of the basic and acidic form protein kinases to phosphatidylserine (PS), diolein, and Ca2+, the basic form was found to contain both regulatory and catalytic domains of PKC, whereas the acidic form was suggested to represent the PKC catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of interferon on phospholipid methylation by peripheral blood mononuclear cells.

The effect of human interferon (IFN) preparations on the metabolic pathway leading to the synthesis of phosphatidylcholine (PC) by a stepwise addition of methyl groups to phosphatidylethanolamine (PE) was investigated in human peripheral blood mononuclear (PBMN) cells. An inhibition of the synthesis of PC via this pathway was regularly observed with both alpha- (recombinant or natural) and beta-IFN. This inhibition was apparent within the first 5 min of treatment, reached its maximum between 15 min and 1 hr, and persisted at the same level until 6 hr, the last time point examined. Each of the transmethylated products of PE underwent a similar inhibition, as measured by the turnover rate of individual products. The intracellular pool of the methyl donors, methionine and S-adenosyl-methionine (SAM), was shown to be unaffected. The methyltransferase activity of IFN-pretreated cell extracts was unchanged. These findings support the hypothesis that IFN induces a functional change in phospholipid methylation at the level of organized membrane-bound phospholipid methyltransferase enzymes in intact cells.

Immunoregulatory role of in vitro differentiated macrophages on human natural killer (NK)-cell activity.

Immunoregulatory effects of human macrophages on natural killer (NK) activity were studied. Monocytes were isolated by adherence to plastic, after leukapheresis of normal blood donors, and cultured for 1 to 14 days. In vitro-differentiated (5-7 days) human macrophages consistently and significantly (P less than 0.01) augmented NK activity of fresh autologous or allogeneic PBMNC. During culture, these macrophages also developed increased antitumor cytostatic activity. The optimal time for both the expression of cytostatic activity and up-regulation of NK activity was 5-7 days in culture. In contrast, 12- to 14-day macrophages significantly suppressed NK activity and had less cytostatic activity. Macrophages in culture demonstrated shifts in Leu-M3+HLA-DR+ phenotype from the mean of 60% +/- 11 (SD) in fresh monocytes to 90% +/- 5 between Days 5 and 7 in culture and then down to 10% +/- 5 in 14-day cultures. The activity of NK (CD56+CD3-) cells, purified by Percoll gradient centrifugation and flow cytometry, was up-regulated directly by in vitro-differentiated macrophages at low macrophage to NK cell ratios, and this up-regulation was not dependent on T lymphocytes or other accessory cells. The modulation of NK activity by differentiated macrophages was not MHC-restricted and depended on the viability and cellular integrity of macrophages. Sonicated macrophages could no longer up-regulate NK activity. This study shows that antitumor effects mediated by human in vitro differentiated LeuM3+HLA-DR+ macrophages may simultaneously involve more than one mechanism, namely direct cytostasis of tumor cells and activation of NK cells.

Differential in vitro modulation of suppressor and antitumor functions of mouse macrophages by lymphokines and/or endotoxin.

Peritoneal macrophages of normal mice exhibited natural suppressor activity, as indicated by their ability to inhibit the proliferation of spleen cells in response to stimulation with phytohemagglutinin (PHA) or concanavalin A (Con A). Their suppressor function could be modulated in vitro with a variety of treatment regimens. High-dose lipopolysaccharide (LPS) (LPSH; 10 micrograms/ml) or lymphokines (supernatant from Con A-stimulated spleen cells) plus low-dose LPS (LPSL; 10 ng/ml) caused a reduction in the suppressor activity of adherent peritoneal macrophages. In contrast, these same treatments induced the macrophages to become tumoricidal and cytostatic for tumor cells, indicating a major dissociation between the regulation of suppressor and cytotoxic activities of macrophages. The lack of correlation between these activities was further demonstrated by macrophages that had been activated in vitro by Corynebacterium parvum: these cells expressed high tumoricidal and cytostatic activities, and also strong suppressor activity. The suppressor function could be selectively downregulated by in vitro pretreatment with LPSH.

Spontaneous cytotoxicity by monocyte-enriched subpopulations of human peripheral blood mononuclear cells against human or mouse anchorage-dependent tumour cell lines. Contribution of NK-like cells.

Human peripheral blood mononuclear cells (PBMNC) were found to be cytotoxic for mouse or human anchorage-dependent target cell lines in a 48-72 h [125I]iododeoxyuridine (IUDR) release assay. Unfractionated, adherent or nonadherent cells had significant levels of cytotoxicity, as did cells fractionated according to size into 'lymphocytes' or 'monocytes' by elutriation. Intermediate size cells, not enriched for monocytes, had high levels of cytotoxicity. In all fractions tested, including adherent populations, some cells with the morphology of large granular cells were observed. Treatment of all fractions with interferon (IFLrA, a purified, recombinant alpha-IFN) boosted cytotoxicity against four target cells lines. Treatment with lymphokines containing putative 'macrophage-activating factor' (MAF) also enhanced cytotoxicity in fractions depleted of monocytes. Culture in fetal bovine serum enhanced cytotoxicity mainly in unfractionated and nonadherent PBMNC. These experiments indicated that NK-like cells can be appreciable contaminants in elutriator-purified monocyte-enriched or adherent cell populations and thereby contribute to observed cytotoxicity, particularly after pretreatment with IFN or other stimulatory factors.